Viruses are ultramicroscopic infectious agents, comprised of a piece of nucleic acid (DNA or RNA) wrapped in a thin coat of protein, which replicate only within cells of living hosts. As many are pathogenic; there is a widespread need to remove viruses from biological products such as fluids, especially for use in the medical and diagnostic field. In addition, the relatively new field of gene therapy, wherein virus is used as carriers of genes intended for therapeutic applications, also requires procedures for the purification of virus. Finally, research directed to the virus vaccine field also requires availability of efficient methods of purifying virus. For example, the recent outbreak of avian flu in some parts of the world resulted in increased research efforts directed to the finding of an efficient vaccine.
WO 9826048 (Schering Corporation) relates to virus purification by anion exchange followed by size exclusion chromatography. More specifically, this patent application discloses a method for purification of a virus preparation comprising:
a) subjecting the virus preparation to anion exchange chromatography, wherein the virus is eluted from an anion-exchange chromatographic medium; and
b) subjecting the virus product of step A to size exclusion chromatography, wherein the virus is eluted from a size exclusion chromatographic medium.
EP 0 848 752 (Aventis Pasteur) relates to purification of viruses by cation and ion exchange chromatography. More specifically, this patent application relates to a method for purifying virus obtained from a cell culture, which consists in separating by ion-exchange chromatography the viruses from the cell proteins and DNA originating from the culture, characterized in that it comprises at least one step of anion exchange chromatography and one step of cation exchange chromatography. In one embodiment, the viruses purified are influenza viruses.
U.S. Pat. No. 6,537,793 (Aventis) relates to viral purification, and more specifically to viral purification on an ion exchanger to which ligands have been coupled to a matrix via extenders. The matrix may be chosen from agarose, dextran, acrylamide, silica and poly[styrene-divinylbenzene], preferably, the matrix consists of agarose. Advantageously, the flexible arm is of a hydrophilic nature and consists of a polymer of synthetic or natural origin. Among the polymers of synthetic origin, polymers consisting of monomers of polyvinyl alcohols, polyacrylamides, polymethacrylamides or polyvinyl ethers are mentioned. By way of polymer of natural origin, in particular polymers of a polysaccharide nature chosen from starch, cellulose, dextran and agarose are suggested. Preferably, the flexible arm is functionalized by grafting a group capable of interacting with an anionic molecule, such as a ternary or quaternary amine. It is stated to be particularly advantageous to use a strong anion exchanger. A support presented as being particularly preferred is Q SEPHAROSE™ XL (GE Healthcare, Uppsala, Sweden), which is an agarose base matrix to which Q groups have been attached via dextran.
WO 97/15661 (Allen) relates to a microporous pathogen-killing composition. More specifically, a microporous substance is disclosed, wherein the pores are sized to permit entry of pathogenic particles but exclude blood cells, and a singlet oxygen-generating system is bound to the microporous substance. In a particular embodiment, a ligand that binds a pathogenic particle is also bound to the microporous substance, which ligand may be selected among high mannose glycans, N-glucosamine, the N-acetylglucosaminyl core of oligosaccharides, the monosyl core of complex-type N-linked glycans, mannan, α-methylmannoside, haloperoxidases, sulphated polysaccharides, low molecular weight dextran sulphate, and lectins. The preferred ligands include the haloperoxidase myeloperoxidase and the lectin concavalin A.
US 2003/0187227 (Lihme) relates to the isolation of immunoglobulins from a solution containing immunoglobulins, e.g. hybridoma cell culture supernatants, animal plasma or sera, and to a solid phase matrix useful in said isolation. More specifically, the solid phase matrix comprises a functionalised matrix backbone carrying a plurality of functional groups of the formula: M-SP1-L, wherein M designates the matrix backbone; SP1 designates a spacer and L designates a ligand comprising a mono- or bicyclic optionally substituted aromatic or heteroaromatic moiety. In one embodiment, the ligand comprises at least one nitrogen, sulphur or phosphorus atom, such as a ring atom or as a substituent on the heteroaromatic ring, such as an amino or nitro group or a sulfonic acid group or a phosphonic acid group.
U.S. Pat. No. 5,447,859 (Prussak) relates to a method for the purification or removal of retroviruses using sulphated cellulose. More specifically, moderately to highly sulphated oligosaccharides are stated to be useful for a variety of purposes, including the purification of viruses, the removal of vital contaminants in preparations, and the delivery of viruses. In this context, “moderately sulfated” refers to oligosaccharides having at least about 6-15 μmoles sulphate per gram of oligosaccharide; and “highly sulfated refers to greater than about 15 μmoles sulphate per gram of oligosaccharide. Generally, in this disclosure, the maximum level of sulfation is stated to be about 20 μmoles per gram. Suitable oligosaccharides include alkyl oligosaccharides, with cellulose, heparin and agarose as preferred oligosaccharides.
U.S. Pat. No. 4,724,210 (Oka et al) relates to a method for the purification of influenza virus, which comprises subjecting a solution containing the influenza virus to column chromatography using, as a gel for chromatography, a sulfuric acid ester of cellulose or a crosslinked polysaccharide. The method can provide highly purified influenza virus which is useful for obtaining an effective vaccine against influenza. The sulfuric acid ester of a crosslinked polysaccharide used includes a sulfuric acid ester of polysaccharide, such as dextran, cellulose, or agarose, which is crosslinked with a crosslinking agent. Further, the gel for chromatography is characterized in that it is prepared by directly sulfating cellulose or a crosslinked polysaccharide, which are water-insoluble, with a sulfating agent such as chlorosulfonic acid or anhydrous sulfuric acid in an organic solvent, e.g. pyridine. Thus, the resultant gel is water-insoluble and highly stable.
However, despite the above-discussed methods, there is an increasing demand for optimized and generic products for the purification of virus, especially for the purification of influenza virus.